Genomic insights reveal community structure and phylogenetic associations of endohyphal bacteria and viruses in fungal endophytes

ABSTRACT Background Endohyphal microbiomes, comprising bacteria and viruses, significantly influence fungal phenotypes, host fitness, and ecological interactions. Endohyphal bacterial symbionts are known to affect fungal pathogenicity, secondary metabolite production, and adaptability, yet many aspects of their diversity and interactions remain uncertain. In contrast, endohyphal viruses, despite their widespread presence, are poorly understood in terms of their diversity, ecological roles, and evolutionary relationships with fungal hosts. These gaps highlight the need for integrative studies to explore the composition, diversity, host associations, and functional roles of both bacterial and viral communities in fungi. This study aimed to (1) characterize the diversity of endohyphal bacterial and viral communities within selected fungal endophytes using genomic and transcriptomic approaches, and (2) test for host specialization through phylogenetic signals and core microbial taxa. Results Endohyphal microbial communities from 19 fungal isolates from eight fungal orders ( Amphisphaeriales, Botryosphaeriales, Diaporthales, Glomerellales, Mucorales, Pleosporales, Sordariales , and Xylariales ) obtained from Fagus grandifolia leaves were characterized. Bacterial communities exhibited high diversity and significant phylogenetic signals, with core taxa such as Bacillales, Burkholderiales, Enterobacterales, Hyphomicrobiales , and Pseudomonadales , shared across all fungal samples. Specific bacterial taxa displayed potential host specialization, including Moraxellales, Sphingomonadales , and Streptosporangiaceae for Amphisphaeriales fungal samples; Enterobacterales (e.g., Enterobacteraceae ), Hyphomicrobiales (e.g., Rhizobiaceae ), and Micrococcales for Glomerellales ; and Cytophagales for Diaporthales . Viral communities were less diverse, with Bamfordvirae and Heunggongvirae (double-stranded DNA viruses) identified as core taxa in metagenomic data, while metatranscriptomic data revealed no core taxa. Surprisingly, only a few reads of double-stranded RNA mycoviruses were detected. Conclusions The findings suggest a level of host specialization in bacterial communities and a more complex, limited association for viral communities, with dsDNA dominating the endohyphal virome. This study enhances our understanding of fungal-microbe interactions and highlights the ecological and evolutionary dynamics of endohyphal microbiota. Future efforts to expand reference databases and investigate the functional roles of these microbial taxa will further elucidate their contributions to fungal biology, impacts on their plant hosts, and ecosystem processes.